This invention relates to the in vitro clinical determination of glycerol esters in body fluids, and more particularly to the hydrolysis of glycerides to glycerol for analysis.
Glyceride concentration, particularly triglycerides in the body fluids of man and other animals, are known to be significant to a number of pathological conditions and its determination is a common clinical procedure. Generally, the glycerides are hydrolyzed and the glycerol thus freed is assayed by a variety of techniques. Hydrolysis by both chemical and enzymatic means are known, with the latter being generally preferred for safety, specificity and convenience. A number of single source lipases have been proposed, for example, Rhizopus arrhizus lipase (hereinafter LIPR) in U.S. Pat. No. 3,759,793, and Pseudomonas fluorescens lipase (hereinafter LPL) in Chimica Chimica Acta, 81 (1977) 125:130. However, such single source lipases either will not fully hydrolyze glycerides or will do so only under limited conditions or with impractically large amounts of enzyme or time. Thus, so far as known, such single source lipases are not used. Instead, mixtures of enzymes are employed, for example, LIPR with protease as disclosed in U.S. Pat. No. 3,708,591; mixtures of LIPR and carboxylesterase as disclosed in U.S. Pat. No. 3,862,009; and mixtures of LIPR with Candida cylindracea as disclosed in U.S. Pat. No. 4,056,442. Such mixtures also have limitations, however. Proteases limit stability of the reagent since they eventually attack protein in the reagent or sample, carboxyleasterases are difficult to purify, and combinations of LIPR and Candida cyclindracea lipases are inhibited by wetting agents commonly used in reagents, samples or controls, especially in automated, continuous flow analyzers.
Automated continuous flow analyzers impose important limitations on conditions for hydrolyzing triglycerides. Frequently calibrators and standards contain surfactants, while use of small samples and short reaction times are common. For example, the Technicon SMAC instrument provides a fixed incubation time of 2.5 minutes at 37.degree. C., and employs surfactants in its calibrating standards which inhibit some lipases, for example the mixture disclosed in U.S. Pat. No. 4,056,442.
Following hydrolysis of triglyercides, the glycerol freed may be assayed by any suitable procedure. The glycerol kinase procedure is commonly used and is described in a number of publications, including U.S. Pat. Nos. 3,703,591; 3,759,793; and 4,056,442 and the Technicon Instruments Corporation publication dated March, 1976, entitled "Technicon Method No. SG4-0023PC6. These disclosures are herein incorporated by reference.
In one procedure, the assay reactions are summarized as follows: ##STR1## As used above, FFA=free fatty acids; ATP=adenosine triphosphate; GK=glycerol kinase; GP=glycerol phosphate; ADP--adenosine diphosphate; PEP=phosphoenolpyruvic acid; LDH=lactate dehydrogenase; PK=pyruvate kinase; NAD and NADH=Nicotinamide adenine dinucleotide, oxidized and reduced. NADH absorbs at 340 nm while NAD does not, the decrease in optical absorbance at 340 nm being measured and calibrated as a measure of the concentration of triglycerides in the sample. Methods which measure the reduction of NAD by other enzymatic means are also known and are described for example in U.S. Pat. No. 4,056,442 and in Clinica Chimica Acta, 81 (1977) 125:130. These disclosures are also incorporated herein by reference.